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| United States Patent |
6,159,717
|
|
Savakis
, et al.
|
December 12, 2000
|
Eukaryotic transposable element
Abstract
Disclosed are isolated transposable elements, or isolated DNA sequences
which encode a transposase protein (or a portion of a transposase
protein). The isolated transposable elements or the isolated DNA sequences
being characterized by the ability to hybridize to the DNA sequence of
Minos-1. The invention also relates to a purified transposase protein, or
peptide fragments thereof, encoded by such DNA sequences. Such
transposable are useful in methods for the stable introduction of a DNA
sequence of interest into a cell. The invention further relates to
transgenic animals produced by such methods. The sequence information
disclosed herein is useful in the design of oligonucleotide primers which
are useful for the isolation of related members of the Tc-1 family of
transposable elements.
| Inventors:
|
Savakis; Charalambos (Heraklion, GR);
Franz; Gerald H. (Romzargasse, AT)
|
| Assignee:
|
Institute for Molecular Biology and Biotechnology/Forth (Crete, GR)
|
| Appl. No.:
|
195726 |
| Filed:
|
November 18, 1998 |
| Current U.S. Class: |
435/194; 435/193; 435/196; 530/350; 536/23.1; 536/23.2; 536/23.5 |
| Intern'l Class: |
C12N 009/12; C12N 009/16 |
| Field of Search: |
530/350
435/196,69.1,193,194
536/23.1,23.2,23.5
|
References Cited
U.S. Patent Documents
| 5348874 | Sep., 1994 | Savakis et al. | 435/196.
|
Other References
Loukeris, T.G. et al., "Introduction of the transposable element Minos into
the germ line of Drosophila melanogaster", Proc. Natl. Acad. Sci. USA,
92:9485-9489, (Oct. 1995).
Loukeris, T.G. et al., "Gene Transfer into the Medfly, Ceratitis capitata,
with a Drosophila hydei, Transposable Element", Science, 270:2002-2005
(Dec. 1995).
"D. hydei Minos transposon-like element encoding transposase", submitted to
EMBL Data Library by Charalambos Savakis; Released by EMBL Data Library on
Sep. 12, 1991.
Franz G., and Savakis C., "Minos, a new transposable element from
Drosophilia hydei, is a member of the Tc1-like family of transposons,"
Nucl. Acids Res., 19(23):6646 (1991).
Franz G., et al., "Mobile Minos elements from Drosophila hydei encode a
two-exon transposase with similarity to the paired DNA-binding domain,"
Proc. Natl. Acad. Sci. USA, 91(11):4746-4750 (1994).
|
Primary Examiner: Nashed; Nashaat T.
Attorney, Agent or Firm: Hamilton, Brook, Smith & Reynolds, P.C.
Parent Case Text
RELATED APPLICATIONS
This application is a divisional of U.S. application Ser. No. 08/530,566,
filed Sep. 20, 1995 (now U.S. Pat. No. 5,840,865), which is a
continuation-in-part of U.S. application Ser. No. 08/239,765, filed May 9,
1994, which is a divisional of U.S. application Ser. No. 07/946,237, filed
Sep. 14, 1992 (now U.S. Pat. Ser. No. 5,348,874), the entire teachings of
which are incorporated herein by reference.
Claims
What is claimed is:
1. A purified transposase protein, or peptide fragments therof having
transposase activity encoded by a DNA sequence characterized by the
ability to hybridize in a buffered solution of 0.9 M NaCl, at a
temperature of 55.degree. C., to DNA having a sequence selected from the
group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,
SEQ ID NO:6 and SEQ ID NO:8.
2. The purified transposase protein of claim 1 encoded by a DNA sequence
selected from the group consisting of: SEQ ID NO:6 and SEQ ID NO:8.
3. A purified transposase protein of claim 2 comprising a protein of about
341 amino acids.
4. A purified transposase protein having an amino acid sequence selected
from the group consisting of: SEQ ID NO:7 and SEQ ID NO:9.
Description
BACKGROUND OF THE INVENTION
The Tc1-like family of transposons and the retroviral-like transposons are
unique for their wide dispersion in diverse organisms. Six members
belonging to the Tc-1-like family have been characterized in nematodes,
diptera and fish: Tc1 in Caenorhabditis elegans, TCb1 in Caenorhabditis
briggsae, HB1 in Drosophila melanogaster, Uhu in Drosophila heteroneura,
Minos in Drosophila hydei and Tes1 in the Pacific hagfish Eptatetrus
stouti. All are characterized by a relative short length (1.6 to 1.8 kb),
the presence of inverted terminal repeats, and significant sequence
similarity in the region between the repeats.
The Minos-1 transposable element has been identified as a 1775 bp dispersed
repetitive sequence inserted within the transcribed spacer in one of the
repeats of Drosophila hydei (Franz and Savakis, Nucl. Acids Res. 19:6646
(Dec. 11, 1991)). The element is characterized by 255-bp long perfect
inverted repeats and the presence of two long, non-overlapping open
reading frames (ORFs) on the same strand. The longest of the ORFs shows
approximately 30% sequence identity with TcA, but does not begin with an
ATG codon. It appears, therefore, that the cloned element represents a
defective member of the Minos family, as is the case with all previously
sequenced Tc1-like elements, with the possible exceptions of Tc1 and TCb1.
SUMMARY OF THE INVENTION
The invention relates to an isolated transposable element, or an isolated
DNA sequence which encodes a transposase protein (or a portion of a
transposase protein). The isolated transposable element or the isolated
DNA sequence is characterized by the ability to hybridize to the DNA
sequence of Minos 1 under stringent hybridization conditions. The
invention also relates to a purified transposase protein, or peptide
fragments thereof, encoded by such DNA sequences.
In another aspect, the invention relates to a method for the stable
introduction of a DNA sequence of interest into a cell. This method
involves the use of an isolated transposable element of the type described
in the preceding paragraph, the isolated transposable element being
modified to include the DNA sequence of interest flanked by the termini of
the isolated transposable element. This modified transposable element is
introduced into the cell in the presence of a transposase protein, or a
DNA sequence encoding a transposase protein. The role of the transposase
protein is to catalyze the transposition of the modified transposable
element containing the DNA sequence of interest into the genomic DNA of
the cell.
In a third aspect, the invention relates to a method for isolating members
of the Tc-1 family of transposable elements from genomic DNA of a
eukaryote of interest. According to this method, oligonucleotide primers
are provided which are complementary to a sequence of at least about 12
consecutive nucleotides which encode amino acids which are highly
conserved in aligned sequences of nematode Tc-1 family members and Minos
family members. These oligonucleotide primers are used to prime
amplification by the polymerase chain reaction (PCR). The amplification
products are then used to isolate DNA encoding the entire Tc-1 family
member from the eukaryote of interest by conventional methods.
In a fourth aspect, the invention relates to a transgenic animal. The
transgenic animal is produced by a method which involves the use of an
isolated transposable element characterized by the ability to hybridize to
the DNA sequence of Minos 1, the isolated transposable element being
modified to include the DNA sequence of interest flanked by the termini of
the isolated transposable element. This modified transposable element is
introduced into a cell in the presence of a transposase protein, or a DNA
sequence encoding a transposase protein.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-1C is a diagram providing the consensus sequence of elements
Minos-1, Minos-2 and Minos-3 with nucleotide deletions after nucleotides
365, 678 and 715. The terminal inverted repeats and the intron sequence
are shown in small letters. Differences between the three elements are
indicated above and below the nucleotide sequence. More specifically,
nucleotide 896 is a G in Minos-2 and Minos-3 and an A in Minos-1.
Nucleotide 1157 is a C in Minos-1 and Minos-3 and a T in Minos-2.
FIGS. 2A-2C is a diagram providing the consensus sequence of elements
Minos-1, Minos-2 and Minos-3. The terminal inverted repeats and the intron
sequence are shown in small letters. The first and last nucleotides of the
sequence, A and T, respectively, are generated by a duplication of the
chromosomal target site TA during insertion of the element. The deduced
amino acid sequence of two open reading frames is shown above the
nucleotide sequence. Differences between the three elements are indicated
above and below the nucleotide sequence. More specifically, nucleotide 900
is a G in Minos-2 and Minos-3 and an A in Minos-1. Nucleotide 1161 is a C
in Minos-1 and Minos-3 and a T in Minos-2. Amino acid residue 148 is a
tryptophan in Minos-2 and Minos-3 and a stop codon in Minos-1. Amino acid
residue 235 is a serine in Minos-1 and Minos-3 and a leucine in Minos-2.
FIG. 3A is a diagram of the insert of the transposon plasmid pMihsCcw. ML
and MR signify the left- and right-end parts of Minos, respectively.
Speckled boxes indicate the D. melanogaster Hsp70 promoter (Hsp70-P) and
terminator (Hsp70-T) sequences. Wide hatched bars indicate the Minos (M)
and Medfly white (W) sequences that were used as probes for the analysis
of transformants.
FIG. 3B is a diagram of the insert of the Minos helper plasmid pHSS6hsMi.
Speckled box indicates the D. melanogaster Hsp70 promoter (Hsp70-P)
sequence. Salient restriction sites are shown. Exon 1 and exon 2 are also
referred to herein as open reading frame 1 (ORF1) and open reading frame 2
(ORF2), respectively. IR indicates the right-hand terminal inverted
repeat.
FIG. 4 is a bar graph depicting the frequencies of transformants among G1
progeny. Bars indicate the numbers of G1 flies from the individual cages.
The sex of the G0 flies in each cage is indicated. The numbers above cages
1, 3, 25 and 33 indicate the w.sup.+ flies that were recovered from these
cages.
SEQUENCE LISTINGS CROSS-REFERENCES
In portions of the Specification, the following sequence listing
cross-reference is applicable:
SEQ ID NO: 1 Nucleic acid sequence of Minos-1 with nucleotide deletions
after nucleotides 365, 678 and 715.
SEQ ID NO: 2 Nucleic acid sequence of Minos-2 with nucleotide deletions
after nucleotides 365, 678 and 715.
SEQ ID NO: 3 Nucleic acid sequence of Minos-3 with nucleotide deletions
after nucleotides 365, 678 and 715.
SEQ ID NO: 4 Nucleic acid sequence of Minos-1.
SEQ ID NO: 5 Deduced amino acid sequence of Minos-1.
SEQ ID NO: 6 Nucleic acid sequence of Minos-2.
SEQ ID NO: 7 Deduced amino acid sequence of Minos-2.
SEQ ID NO: 8 Nucleic acid sequence of Minos-3.
SEQ ID NO: 9 Deduced amino acid sequence of Minos-3.
SEQ ID NO: 10 MVWGC.
SEQ ID NO: 11 WPSQSPDL.
SEQ ID NO: 12 WPSNSPDL.
DETAILED DESCRIPTION OF THE INVENTION
The invention disclosed herein is based on the initial discovery of
Minos-1, an apparently defective member of the Tc-1 family of transposable
elements. This 1779-bp element is characterized by perfect inverted
repeats of 255-bp at each termini. The sequence encodes two
non-overlapping reading frames, one of which has significant similarity
with the putative transposase encoded by the transposable element Tc1 of
Caenorhabiditis elegans. However, the Minos-1 element, because of a stop
codon within the putative transposase gene, apparently cannot encode an
active transposase.
In an effort to identify sequences related to the Minos-1 sequence, genomic
DNA of D. hydei was probed with a portion of the Minos-1 sequence under
stringent hybridization conditions. As discussed in detail in the
Exemplification section which follows, two full-length related sequences
were identified, both of which encode an active transposase.
Isolated Nucleic Acids and Uses Thereof
Thus, in one aspect, the subject invention relates to an isolated
transposable element which hybridizes to the DNA sequence of Minos-1 under
stringent hybridization conditions. As used herein, stringent
hybridization conditions are considered to be hybridization in a buffered
solution of 0.9 M NaCl at 55.degree. C. In D. hydei there are up to
30-copies detected which hybridize to Minos thus, it is likely that a
large number of variants can be isolated using these conditions.
Comparable hybridization stringency can be established at other salt
concentrations and temperatures. This is accomplished, for example, by the
inclusion of organic denaturants such as formamide in the hybridization
buffer. DNA sequences which hybridize to the Minos-1 sequence under
stringent hybridization conditions are referred to herein as members of
the Minos family of transposable elements. DNA sequences which hybridize
to the Minos-1 sequence under stringent hybridization conditions include,
for example, the Minos-2 and Minos-3 DNA sequences. Other examples of DNA
sequences which hybridize to the Minos-1 sequence under stringent
hybridization conditions include Minos-1, Minos-2 and Minos-3 DNA
sequences having base deletions, insertions and/or substitutions.
The term transposable element, as used herein, refers to a DNA sequence
whose excision from/insertion into genomic DNA is catalyzed by a
functional transposase protein encoded by a non-defective member of the
Minos family of transposable elements. A member of the Minos family which
encodes a functional transposase and possesses other necessary cis-acting
elements (e.g., inverted terminal repeats) falls within this definition.
In addition, a transposable element which encodes a defective transposase
(e.g., Minos-1 itself) falls within this definition. As discussed in
greater detail below, such defective transposable elements can be used in
conjunction with a helper element (i.e., a member of the Minos family
which encodes a functional transposase) to introduce a DNA sequence of
interest into a cell (e.g, a eukaryotic cell such as an animal, plant or
yeast cell or a prokaryotic cell such as a bacterial cell).
The invention also relates to an isolated DNA sequence encoding a
functional transposase protein, or a portion of a transposase protein,
encoded by a member of the Minos family. Such a DNA sequence need not
retain the ability to transpose in the presence of the encoded transposase
protein. A sequence encoding a functional transposase protein can be used
to prepare an expression construct which can be used to produce the
transposase protein by recombinant DNA methodology. Such a recombinant
protein can be over-produced in a eukaryotic (e.g., yeast) or prokaryotic
host cell (e.g., E. coli), and subsequently purified by conventional
methods.
The active transposase can be used in a variety of ways. For example, as
discussed below, the transposase can be co-introduced into a eukaryotic
cell with a modified transposon carrying a DNA sequence of interest to
catalyze the insertion of the modified transposon into the genomic DNA of
the eukaryotic cell. This is an alternative to the co-introduction of a
helper construct in eukaryotic cells which do not constitutively produce
the Minos transposase.
In addition, the transposase, or portions thereof, can be used to produce
antibodies (monoclonal and polyclonal) reactive with the transposase
protein. Methods for the production of monoclonal and polyclonal
antibodies are straightforward once a purified antigen is available.
Through the isolation and DNA sequence analysis of additional members of
the Minos family, refinement of the consensus sequence of FIGS. 2A-2C is
possible. This refined consensus sequence can be used to predict
modifications of the transposase protein which will affect the specific
activity of the transposase. Such predictions are easily tested by
modifying the DNA sequence of an expression construct encoding the
transposase by site-directed mutagenesis to either bring the sequence into
a greater degree of conformance with the consensus sequence, or a lesser
degree of conformance with the consensus sequence. The affect of such
changes on the activity of the transposase protein are monitored by
assessing the affect of the mutation on transposition frequency catalyzed
by the recombinant transposase.
Methods for the Introduction of DNA Sequences into a Cell
Transposable elements of the Minos family, and the active transposase
encoded by such elements, are useful in methods for introducing a DNA
sequence of interest into a cell (e.g., a eukaryotic cell such as an
animal, plant or yeast cell or a prokaryotic cell such as a bacterial
cell). Typically, the DNA sequence of interest will be a gene which
encodes a protein. Such a gene can be placed under the regulatory control
of a promoter which can be induced or repressed, thereby offering a
greater degree of control with respect to the level of the protein in the
cell. In addition to a DNA sequence encoding a protein, any other DNA
sequence can be introduced by this method including, for example,
regulatory sequences.
The Minos transposable elements can be used to introduce a DNA sequence of
interest into the cells of invertebrates. For example, the Minos
transposable elements can be used to introduce a DNA sequence of interest
into the cells of arthropods. Arthropods include, for example,
crustaceans, arachnids, myriapods and insects.
The Minos transposable elements can be used to introduce a DNA sequence of
interest into either germ line or somatic cells. The introduction of DNA
into germ line cells has the significant advantage that the DNA sequence
of interest will be contained in all cells of the mature organism and
transmitted to progeny.
The Minos transposable element has been demonstrated to function in a
species which is separated from the Minos source species by an
evolutionary distance of 40 million years. This represents the first
demonstration of a mobile element which can function autonomously in the
germ line of eukaryotes separated by such an evolutionary distance and is
likely to lead to the development of a long-sought transformation system
applicable across taxonomic barriers.
However, even within the dipteran class, significant important applications
for the Minos element exist. Listed below are examples of a variety of
plant and animal pests, and human disease vectors which fall within the
dipteran genus.
______________________________________
COMMON NAME
______________________________________
AGRICULTURAL PESTS
Ceratitis capitata Medfly
Anastrepha species Carribean fruit fly
Dacus oleae Dacus
Bactrocere species Oriental fruit fly
ANIMAL PESTS
Cochliomya hominivorax
Screw Worm Fly
Lucilia cuprina Sheep blowfly
Simulium species Black fly
HUMAN DISEASE VECTORS
Anopheles species mosquito
Aedes species mosquito
Musca domestica house fly
______________________________________
Methods currently employed to control the populations of certain members of
the dipteran class include the release of sterile males. An example of the
utility of the germ line transformation methods of this invention includes
the improvement of the existing release method. The methods of this
invention can be used to improve such methods by enabling sexing schemes
and for developing strains with desired characteristics (e.g., improved
viability in the field), conditional lethal genes for improved safety, and
visible or molecular genetic markers for monitoring. Genetic sexing, i.e.
the capability of selectively killing the females (or transforming them
into males) in mass-rearing facilities, is recognized as an important need
presently. Rearing and releasing only males has several advantages
including lower breeding cost and the avoidance of population explosions
due to inadvertent release of non-sterilized insects.
For example, the Mediterranean fruit fly (Medfly) Ceratitis (C.) capitata
is a major agricultural pest for many fruit species that is geographically
widespread in tropical and temperate regions. The Medfly has been
introduced relatively recently into the New World, and appears to be
spreading rapidly, threatening fruit producing areas in North America
(Carey, J. R., Science, 253:1369 (1991)). Since the mid 1970's, the
sterile insect technique has been used successfully for Medfly eradication
and control. This method relies on the decrease in or collapse of fly
populations following releases of large numbers of sterile insects over
infested areas, and offers an environmentally attractive alternative to
massive spraying with insecticides (Knipling, E. F., Science, 130:902
(1959)). The germ line transformation methods of this invention can be
used to improve the sterile insect technique by, for example, enabling
sexing schemes. The germ line transformation methods of this invention can
also be used for developing Medfly strains with desired visible markers
that can be used for monitoring effective population control.
The methods are also useful for insects for which it might be desirable to
introduce new traits in the genetic pool, rather than controlling the
population levels. For example, the presence of several sympatric
sub-species of Anopheles gambiae, all of which transmit malaria, makes it
highly unlikely that population control with biological methods such as
the sterile insect technique will work. An alternative scheme might
involve spreading genes for refractoriness to parasite infection into the
existing populations of Anopheles through the use of transposable
elements. Population dynamics simulations indicate that this can be
effected by releasing relatively small numbers of individuals carrying an
autonomously transposing element.
The element may be actively transposing in other taxa (e.g. vertebrates)
under the appropriate conditions thus, it will be recognized by those
skilled in the art that the methods disclosed herein relating to diptera
can be extended to higher eukaryotes. If the transposase is functional
when expressed or otherwise introduced in vertebrate embryos or cells, it
is possible to develop transformation methods based on Minos elements for
non-insect species as well.
A transposon-based method for producing transgenic animals or for stably
transfecting cells in vitro has very important advantages compared to the
methodology presently used. For example, stable integration of DNA into
the germline of several mammals is now routinely achieved by
micro-injecting linear DNA molecules into the nucleus of early embryos.
Some of the animals that develop from injected embryos are mosaics for
integration events and in only a fraction of these the germ line is
involved. Moreover, most events consist of integration of tandem repeats
of the injected DNA; single-insertion events do occur at higher
frequencies relative to tandem insertions if DNA is injected at lower
concentrations, but at a considerable cost in time and expense because the
overall transformation frequencies drop.
Using a defined transposon-transposase system may overcome some or all of
these problems. First, as in Drosophila, it may not be necessary to have
to inject the DNA into the nucleus. If a mixture of transposon plus helper
plasmids (or transposon plus purified transposase) is active when
introduced into the cytoplasm, it may be possible to replace costly and
time-consuming micro-injection with other methods, such as use of
liposomes. Second, by controlling the relative transposon/transposase
levels it may be possible to improve the overall efficiency, with a
parallel increase of the frequency of single-insertion events.
Methods for the introduction of the Minos transposon into germ line cells
of diptera are analogous to those previously used in connection with other
transposable elements (see, e.g., Drosophila, A Laboratory Handbook,
Ashburner, M., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y. (1989)). Briefly, the most common approach is to employ a
carrier/helper transposon system. The carrier transposon is a Minos
transposon which has been modified by the insertion of a DNA sequence of
interest in the region of the transposon flanked by the inverted terminal
repeats. Typically, sequences relating to the transposase function are
deleted in order to accommodate the DNA of interest. The helper transposon
is a Minos transposable element which encodes an active transposase. The
transposase catalyzes the transposition of the carrier transposon into the
genomic DNA of the germ line eukaryotic cells. Typically, the helper and
carrier are microinjected into the posterior pole of pre-blastoderm
embryos, where the precursor cells of the germ line develop.
An alternative to the helper/carrier system involves the purification of
active transposase (for example, from an E. coli culture transformed with
a recombinant construct encoding the Minos transposase). The purified
transposase can be co-injected into appropriately selected cells along
with a carrier transposon to effect integration of the carrier into the
recipient genome.
The compositions and methods of this invention are also useful for the
introduction of a DNA sequence of interest into mammalian somatic cells.
Typically this is accomplished in a manner analogous to the methods
described in connection with germ line cells (e.g., helper/carrier systems
are employed). Somatic cell introduction is typically carried out using
cells grown in culture and DNA can be introduced, for example, by calcium
co-precipitation or other conventional methods.
Methods for Isolating Additional TC-1 Family Members
DNA sequence analysis of the members of the Minos family disclosed herein,
and comparison of this sequence information to the sequences of Tc-1
family members from evolutionarily distant organisms (e.g., nematode),
reveal short stretches of conserved amino acid sequence within the
transposase coding region. This high degree of conservation suggests a
method for isolating Tc-1 family members from diverse eukaryotic species.
This method involves the amplification of DNA by polymerase chain reaction
from a eukaryote of interest using primers which are complementary to a
sequence of at least about 12 consecutive nucleotides which encode amino
acids which are highly conserved in aligned sequences of nematode Tc-1
family members and dipteran Minos family members. Such amino acid
sequences include, for example, MVWGC (SEQ ID NO:10), WPSQSPDL (SEQ ID
NO:11) and WPSNSPDL (SEQ ID NO:12).
Exemplification
Materials and Methods
Fly Strains
Standard procedures were used for culturing of Drosophila hydei. All
strains used in this study have been used previously for rDNA work and are
named for the X and Y chromosomes. Strain bb.sup.1 (bb.sup.1 /bb.sup.1
.times.bb.sup.1 /Y) carries a bobbed X chromosome; strain X.sup.7 (X.sup.7
/X.sup.7 .times.X.sup.7 /Y) is a subline of the Dusseldorf wild-type
strain; strain X X/Y(X X/Y.times.X/Y) females carry a compound X
chromosome which has no rDNA. Strain wm1/Y (wm1/Y.times.X-3/Y) females
have a compound X chromosome (wm1); males carry a X-autosome 3
translocation which has no rDNA.
DNA Manipulations and Sequencing
All basic procedures were carried out essentially as described (Maniatis et
al. 1982). DNA from adult females of strain bb.sup.1 was partially
digested with EcoRI and cloned into phage vector .lambda.gt7. To recover
new Minos elements, the library was screened by hybridization with a 1.7
kb HhaI fragment which contains most of the Minos-1 sequence. For
sequencing, the appropriate restriction fragments from positive clones
were subcloned into plasmid vectors pUC8 and pUC9 and nested deletions
were generated by digestion with exonuclease Bal31 followed by subcloning.
Sequencing was performed by conventional methods. Both strands were
sequenced, with a minimum of two independent sequences for each base pair.
Sequence Analysis
Database searches and sequence analysis and manipulations were performed
using programs FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci. USA,
85:2444-2448 (1988)). BLAST (Altschul et al., J. Mol. Biol., 215:403-410
(1990)) and the computer package GCG (Devereux et al., Nuc. Acids Res.,
12:387-395 (1984)). The program CLUSTAL (Higgins and Sharp, 1988) was used
for protein sequence alignments.
Results
The Sequence of Minos
Three new representatives of the Minos family of transposable elements have
been cloned and sequenced; they have been named Minos-2, Minos-3 and
Minos-4, Minos-1 being the element reported previously. Minos-2 and
Minos-3 are complete elements distinct from Minos-1, as judged from the
restriction maps of the flanking DNA and the flanking sequences. The
sequences of the elements, summarized in FIGS. 2A-2C, show very little
variation, differing in only two positions. At position 900 of the
sequence, Minos-2 and Minos-3 have a G instead of the A found in Minos-1.
This transition changes a TAG stop codon to TGG and restores a 603 bp ORF
beginning with ATG at position 878. The second difference is at nucleotide
1161, which is a C in Minos-1 and Minos-3 and a T in Minos-2. This causes
a ser.fwdarw.leu substitution in ORF2 of Minos-2, relative to Minos-1 and
Minos-3. Minos-2 and Minos-3, therefore, have two complete ORFs beginning
with an ATG; ORF1, which can encode a 133 amino-acid peptide, and ORF2,
which can encode a 201 amino-acid peptide.
The Minos-4 clone does not contain a complete element. The sequence of the
cloned DNA fragment begins at the EcoRI site found at position 1172 of the
other members and is identical to the Minos-1 sequence to base 1779.
Apparently Minos-4 represents a partial isolate rather than a defective
member of the family, since the library from which it was isolated was
from DNA cut with EcoRI.
The DNA sequence flanking the cloned elements are different from each
other; this indicates that these elements are inserted at different sites
of the D. hydei genome, and are, therefore, distinct. These sequences are
mainly characterized by a high A/T content, and do not show any other
obvious similarity. In all cases, the inverted repeats end with the
dinucleotide TA, which is at the same time a direct and an inverted
repeat. Because of this, there is some ambiguity in defining the ends of
the element precisely. Shown below are the sequences of the Minos 1-4
insertions sites. The rDNA sequences flanking the Minos elements are shown
in lower case and Minos sequences are shown in upper case. The rDNA
sequence identical to the flanking DNA of Minos-1 has been aligned with
the Minos-1 insertion sequence. It is noted that since gapped sequences
are treated as separate sequences for purposes of the Rules of Practice in
Patent Cases (37 CFR 1.822(o)), and since each of the separate sequences
contain less than 10 nucleotides, the sequences shown below have not been
listed in the Sequence Listing.
In the case of Minos-1, which is inserted into a region which has been
previously sequenced, the external transcribed spacer of the rDNA repeat,
there are two possibilities. As shown below, deleting the sequence which
begins with ACGA and end with TCGT would restore the rDNA sequence; the
element, with an A and a T at the two ends may have inserted between a T
and an A. In this possibility, the element would be 1779 bp long with 255
bp inverted repeats. Alternatively, the element may begin and end with CGA
. . . TCG and produce a target site duplication, as happens with many
other mobile elements. In this possibility the target site duplication
would involve the dinucleotide TA, and the size of the element would be
1777 bp. For numbering, the A of the TA repeat has been designated
nucleotide number 1 of the Minos-1-3 sequences.
rDNA ataat--------------------attaa
Minos-1 ataatACGA------------TCGTattaa
Minos-2 aatatACGA------------TCGTataat
Minos-3 gctttACGA------------TCGTagaag
Minos-4 tttctACGA
.vertline. .vertline.
1775 1
Mobility and Homogeneity of Minos Elements
The striking degree of sequence conservation among the cloned Minos
elements suggests that, as in the case of Tc1, all Minos elements may be
highly homogeneous. To test this the single HhaI site within each of the
terminal repeats of Minos was exploited. The 1.68 kB HhaI fragment of
Minos-1 was used as probe in a Southern blot of genomic DNA from the same
strains, digested with CfoI, an isoschisomer of HhaI. A single, strong
band of approximately 1.7 kb was detectable in all lanes, indicating that
no major deletions or rearrangements are present in the Minos elements
present in these strains.
Comparison of the Proteins Encoded by TC-1 and Minos
The deduced 201 amino acid sequence of the ORF2 in Minos-2 and Minos-3
shows significant sequence similarity with the 201 carboxy terminal
residues of TcA, the putative transposase of Tc1; alignment of the
sequences gives 63 identities (31%) and 91 conservative substitutions
(45%) with only two single-residue insertion-deletions. The two sequences,
however, differ in size; TcA has 72 additional amino acids at the amino
end. The 50 amino-terminal residues of TcA show weak but significant
sequence similarity with the carboxy terminus of Minos ORF2; introduction
of a 60-bp deletion in the Minos DNA sequence creates a long open reading
frame which contains most of ORF1 (codons 1 to 138) and the entire ORF2
extended by 22 codons upstream of the ATG. Interestingly, this 60-bp
sequence, from base 752 to base 811 of the Minos sequence, exhibits
features of an intron. More specifically, the 5' and 3' ends conform to
the consensus splice donor and acceptor sites and a version of the
internal splice signal consensus is found 30 nucleotides upstream from the
3' end.
Divergence of the TCA-Related Sequences
Although Minos inhabits a Drosophila species, it is not more related to the
other Tc1-like elements from Drosophila species, HB1 and Uhu. These
elements, or at least the members which have been sequenced, do not
contain open reading frames comparable in length to that of Tc1. However,
if small numbers of deletions and insertions are introduced in their DNA
sequences, open reading frames can be generated which show significantly
similarity with the TcA sequence. Most of these insertion-deletion changes
involve one nucleotide, presumably representing mutations which have
accumulated in these inactive elements. Table 1 shows a similarity matrix
between the three Drosophila and the two nematode elements, in the regions
corresponding to the hypothetical Minos exon 2. In Table 1, percent
identities are shown above the diagonal; identical/total positions are
shown below the diagonal. Minos shows approximately the same degree of
similarity (between 28 and 36 percent identity) with all the other
elements; HB1 and Uhu show comparable similarities. In a multiple sequence
alignment of the same regions, 21 of the resulting 225 positions (9%) are
invariant and 49 positions (22%) are occupied by related amino acids. It
should also be noted that the similarity between HB1 and Uhu with Tc1 and
Minos extends another 18 codons upstream from the position corresponding
to the first codon of the hypothetical exon 2 of Minos. No other
significant similarities can be detected between Tc1, Uhu, HB1 and Minos
in the sequences between the terminal repeats.
TABLE 1
______________________________________
TC1 TCb1 Minos Uhu HB1
______________________________________
Tc1 71 31 44 33
TCb1 160/223 34 41 35
Minos 70/221 75/222 36 28
Uhu 96/217 89/217 78/218 31
HB1 73/223 79/223 62/222 68/219
______________________________________
The ORF1 Sequence is Related to the Paired Box Sequence
Searches of the nucleic acid and protein sequence data libraries with the
ORF1 sequence using the FASTA and WORDSEARCH algorithms gave no
significant matches. However, the Basic Local Alignment Search Tool
program revealed a similarity with the paired box sequence, a peptide
sequence found in the Drosophila paired gene product, and conserved in
other Drosophila and mammalian genes. This similarity extends
approximately between residues 1 to 96 of the Minos sequence, and residues
35 to 131 of the Drosophila paired protein. Alignment of the Minos
sequence with the Drosophila and human paired box sequences for maximum
similarity shows 16 invariant positions in this region (17%) and 49
positions occupied by related amino acids (51%). The corresponding values
for the human and Drosophila paired sequences are 72% identities and 23%
conserved positions.
Although the Minos-paired similarity is weak compared to that between the
Drosophila and human paired sequences, it is statistically significant.
The similarity scores between the Minos sequence (amino acids 1 to 118 of
ORF1) to the corresponding human paired sequence (amino acids 17 to 135 of
the published sequence) is approximately 10 standard deviations higher
than the average of the scores obtained from 50 comparisons made between
the Minos sequence and 50 randomly shuffled human paired sequences.
Transposition in D. Melanogaster
A D. melanogaster "helper" strain which can overproduce the Minos
transposase upon exposure to heat shock was constructed. The strain was
constructed by introducing a modified Minos element into the germ line by
conventional P element transformation (see, e.g., Drosophila, A Laboratory
Handbook, Ashburner, M., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. (1989)). To place the Minos transposase under heat shock
control, the left-hand terminal repeat of Minos-2 was replaced by the D.
melanogaster hsp 70 promoter. This modified element was inserted into the
P element transformation vector pDM30, which contains a wild-type copy of
the Drosophila rosy (ry) gene as a dominant visible marker. The plasmid
(pPhsM2) was injected into pre-blastoderm embryos of a ry strain, injected
GO adults were mated to ry flies and ry.sup.+ G1 progeny were bred
further. Three independent transformants were recovered, two on the third
chromosome (named M46 and M67) and one on the X (M84). Southern blots
using ry and Minos probes indicated that each of the three transformants
contains a single insertion of the complete sequence between the P element
ends. Northern blots of total RNA from adult transformed flies subjected
to a heat shock showed abundant transcripts hybridizing to Minos probes.
No Minos-related transcripts have been detected by the same probes in RNA
from non-heat shocked flies. The structure of the RNA transcripts was
investigated in another series of experiments discussed below.
Breeding of these transformants showed that they are all homozygous lethal.
This observation was unexpected; the recovery of recessive lethal
mutations due to insertional inactivation of essential genes is a rather
uncommon event in P transformation experiments. Moreover, the insertion
into the X clearly has not caused a "knock-out" mutation since hemizygous
males are viable and fertile; only homozygous females are inviable. This
behavior suggested that the lethality may be dosage- or pairing-dependent;
the latter being more likely because double heterozygotes of the two
insertions in the 3rd chromosome are viable. The observed lethality is a
useful feature which enables one to follow the segregation of the "helper"
chromosomes by keeping them over genetically marked balancers.
Strong evidence for Minos transposition in the germ line was obtained by
first introducing the M67 chromosome into a white background (y,w;
TM3/M67). Pre-blastoderm embryos were injected with a plasmid (pM2w)
containing a complete Minos-2 element with a wild-type copy of the white
(w) gene inserted into its unique EcoRI restriction site within ORF2. The
inserted w sequences provide a dominant selectable marker; in addition
they interrupt ORF2, making the production of active transposase from this
construct highly improbable. Three separate experiments were conducted: In
experiment A injected embryos and the developing larvae and adults were
kept at 18.degree. C., in experiment B they were kept at 25.degree. C.
throughout development, and in experiment C the embryos were subjected to
a 1-hour 37.degree. C. heat shock three hours after injection. All
emerging GO flies (63, 38 and 61, from experiments A, B and C,
respectively) were mated to y,w; TM3/Dgl3 flies and the progeny were
scored for the appearance of the w.sup.+ phenotype. To date, at least
four independent germ line transformation events have been detected in
experiments A and B. Two of these events come from a single GO male from
experiment A and at least two have been recovered from two different GO
flies from experiment B. The results are shown in Table 2 below:
TABLE 2
______________________________________
Insertion
Experiment
GO #G1 Scored w.sup.+ Chromosome
______________________________________
A A10 286 A10.1 X
A10.2 3
A10.3 3
A10.4 --
A10.5 --
A10.6 --
B B13 75 B13.1-3 --
C B33 116 B33.1-18
--
______________________________________
Evidence that the Minos-w.sup.+ transposon can be mobilized in the soma of
flies which produce the transposase has been obtained. Larvae of the
constitution y,w; TM3/[M2w]M67 (progeny of the A10.2 fly), which contain
both transposon and helper sequences, were subjected to heat shock and
adult flies were examined for the appearance of eye color mosaicism. More
than 50% of the flies showed mosaicism of different degrees. Patches of
ommatidia with either reduced or increased pigmentation were observed
which is consistent with the expected result of a somatic deletion or
transposition event. No mosaicism has been detected in flies not subjected
to a heat shock at the larval stage. The somatic instability results
clearly indicate that the w.sup.+ insertions are minos-mediated.
Ananysis of Minos mRNA Transcripts
Total RNA was isolated from the M67 strain, the construction of which is
described above. The structure of mRNA transcripts was investigated by the
polymerase chain reaction (PCR) method of DNA amplification. A
particularly important aspect of this investigation was to determine the
status of the 60 base pair putative intron region (discussed above) in the
mRNA transcripts. As was mentioned previously, this sequence is
characterized by 5' and 3' ends which conform to the consensus splice
donor and acceptor sites, and has a version of the internal splice signal
consensus sequence 30 nucleotides upstream from the 3' end.
To determine the status of this putative intron, PCR priming sites were
selected from exon sequences (ORF1 and ORF2) flanking the putative intron.
The PCR product synthesized in this reaction was cloned and sequenced by
conventional methods. The sequencing experiments revealed unambiguously
that the 60 base pair intron sequence was, in fact, absent in the
amplified DNA.
The removal of the 60-bp sequence in the correctly spliced primary
transcript initiating upstream from ORF1, results in the generation of a
1023-bp open reading frame which encodes a peptide of 341 amino acids. An
alignment of the 273 carboxy-terminal amino acids of this peptide with the
sequences of TcA and the 273-residue hypothetical peptide of TCb1 was
generated by the multiple alignment program CLUSTAL, which introduces gaps
in the sequences to achieve maximum sequence similarity. The three
sequences were aligned without the need of any insertions-deletions (with
the exception of the two one-residue gaps required for optimal alignment
in the ORF2 region) and show an overall 28% identity, i.e. 76 of the 273
positions are invariant. In the region upstream from the first methionine
of ORF2, twelve out of seventy two positions (16%) are invariant; 29
positions (40%) are occupied by structurally related amino acid residues.
Although this degree of similarity is lower than that in the ORF2 region,
it is statistically significant.
The sequence similarity between TcA and the carboxy end of the Minos
hypothetical protein is also reflected in their secondary structures.
Comparisons of .alpha.-helix and .beta.-sheet predictions and
hydrophobicity profiles between the Tcl and Minos sequence show
similarities in several regions. Another feature of the sequences is their
high content, approximately 20%, in basic amino acids. TcA has 29
arginines, 16 lysines and 11 histidines, and the TcA-related Minos
sequence has 20 arginines, 32 lysines and 4 histidines. These are more
abundant at the amino-terminal half of both sequences, although the
position of most is not strictly conserved. The proteins are fairly basic,
with computed isoelectric points of 11.27 for TcA and 10.73 for the
related Minos peptide. The computed pI of the complete hypothetical 361
amino acid Minos protein is 10.97.
Gene Transfer into C. capitata Using Minos Transposable Elements.
Single copies of exogenous DNA can be introduced into the genome of C.
capitata by using a germ line transformation system which utilizes the
transposable element Minos to mediate precise integration of DNA at
acceptable frequencies.
To provide an effective dominant selectable marker for detection of
transformants, an approximately 3.7 kb NotI fragment containing the
wild-type white cDNA of C. capitata, flanked by the D. melanogaster hsp 70
promoter and terminator sequences, was inserted into the NotI site of the
Minos vector pMiNot which was constructed by replacing a 644 bp MscI
fragment of the Minos transposase gene (nucleotides 618 to 1264 of FIGS.
2A-2C) with a NotI linker. This modified element (shown in FIG. 3A) was
inserted into the E. coli vector pTZ18R (Pharmacia), creating a plasmid
(pMihsCcw) having a wild-type copy of the C. capitata white (w) gene as a
dominant visible marker.
To place the Minos transposase under heat shock control, the left-hand
terminal repeat of Minos-2 was replaced by a 456-bp fragment containing
the D. melanogaster hsp 70 promoter. This modified element (shown in FIG.
3B) was inserted into the E. coli vector pTZ18R (Pharmacia), creating the
transposase-producing plasmid pHSS6hsMi.
The plasmids pMihsCcw and pHSS6hsMi were introduced into pre-blastoderm
Medfly w/w embryos by a microinjection procedure similar to that used for
Drosophila. For egg collecting, flies were mass-reared in population cages
at 24.degree. C. Eggs were collected at 24.degree. C. for 60 minutes, and
then were dechorionated, desiccated and microinjected at 18.degree. C.
with a mixture of 100 mg/ml helper and 400 mg/ml transposon plasmid DNA as
described for Drosophila embryos (Rubin, G. M. and Spradling, A. C.,
Science, 218:348 (1982)). Modifications of the procedure were not
necessary, because the eggs of the two species are similar in morphology
and in resistance to desiccation.
A total of 3,998 embryos were injected. After injection, they were left to
hatch under halocarbon oil, and first instar larvae were transferred to
Petri dishes containing standard larval food (Mintzas, A. C. et al., Dev.
Biol., 95:492 (1983)). The 390 adults (G0 generation) resulting from
injected embryos were collected within 12 hours after eclosion and
back-crossed to w flies in small groups consisting of either 5 G0 males
and 10 virgin w females, or 10 G0 females and 5 w males. Fifty-nine such
G0 groups were reared in small plastic cages and the G1 progeny were
collected and handled separately for each group. To induce expression of
the w mini-gene from the Hsp70 promoter, G1 pupae were exposed daily to a
39.degree. C. heat shock for one hour. The 62,510 G1 flies that were
produced were screened for the presence of non-white eye phenotypes. As
shown in FIG. 4, a total of 72 flies with colored eyes were recovered from
four different cages.
The w mini-gene gives partial reversion of the phenotype. Eye color varies
in strength among different transformants. The phenotype is
dosage-dependent with homozygotes having stronger colors than
heterozygotes. These characteristics of w markers are useful in sorting
multiple insertions and in distinguishing homozygous from heterozygous
transformants. The characteristics are due to low levels of expression
combined with chromosomal position effects and have been observed
previously in Drosophila.
To establish transformed lines, individual G1's were initially back-crossed
to w flies. Single pairs of transformed G2 progeny were then mated, and
their homozygous G3 progeny, recognized by their stronger w.sup.+
phenotypes, were used to construct homozygous lines. Table 3 shows the
results from the G1 back-crosses. In these crosses, the non-white eye
(w.sup.+) phenotype was inherited as a single, dominant trait.
To determine the effect of temperature on the expression of the w
mini-gene, a number of G2 pupae were not subjected to the heat shock
treatment. When compared to the heat-shocked cohort, G2 flies which had
not been heat shocked as pupae showed either paler eye color or no eye
color at all; the only exception was lines 3.1 and 3.3, which exhibited an
invariant strong yellow eye phenotype. The heat shock dependence clearly
showed that the flies (perhaps with the exception of 3.1 and 3.3) were
true transformants, rather than revertants of the w mutation.
In cages 3 and 25, differences in the eye color phenotypes of individual
G1's from the same cage were detected and bred true, suggesting that
independent transformation events had occurred in the same cage.
TABLE 3
______________________________________
With heat shock
Without heat shock
Eye color non- non- Eye color
of hetero-
white white white white of homo-
G1 zygotes eyes eyes eyes eyes zygotes
______________________________________
1.1 pale yellow
46 53 0 59 apricot
1.8 pale yellow
220 274 0 77 apricot
1.12 pale yellow
94 69 0 8 apricot
3.1 yellow 267 237 110 97 yellow
3.3 yellow 225 214 53 49 yellow
3.2 pale yellow
132 118 0 76 apricot
3.6 pale yellow
70 81 0 81 apricot
25.7 pale 119 156 116* 91 apricot
apricot
25.8 pink 24 18 0 27 peach
25.9 pink 30 34 0 9 peach
33.2 pale orange
42 50 ND ND orange
33.3 pale orange
29 31 ND ND orange
33.4 pale orange
16 15 ND ND orange
______________________________________
*Eye color much weaker than with heat shock.
To determine the nature of the integration events, DNA from transformants
was analyzed by Southern blot hybridizations using several restriction
enzymes and two probes (see FIG. 3A), one (M) containing the Minos
sequences at the ends of the transposon (which are not present in
non-transformed Medfly), and another (W) containing an internal fragment
of the w cDNA sequences (which is present in the endogenous w gene).
Adult genomic DNA (approximately 10 .mu.g per lane) was digested with a
restriction endonuclease, subjected to agarose gel electrophoresis,
blotted onto nitrocellulose membrane filters and hybridized with .sup.32
P-labeled probes. Membranes were pre-hybridized for 6 hours at 65.degree.
C. in 7% SDS, 0.5 M phosphate buffer pH 7.4, 1 mM EDTA. Hybridization was
for 12-14 hours at 65.degree. C. in 7% SDS, 0.5 M phosphate buffer pH 7.4,
1 mM EDTA. Excess probe was removed by two 10-minute washes with 5% SDS,
40 mM phosphate buffer pH 7.4, 1 mM EDTA at 65.degree. C. followed by a
20-minute wash at room temperature with the same buffer pre-warmed at
65.degree. C.
DNA from lines 3.1, 3.2, 3.3 and 3.6 was cut with SalI and hybridized with
a 1 kb HhaI fragment containing Minos sequences present in pMiNot (M probe
of FIG. 3A).
DNA from the recipient w strain and from lines 3.1, 3.2, 3.3 and 3.6 was
cut with HincII, and probed with a SalI/XhoI fragment containing 1.5 kb of
Medfly w cDNA sequences (W probe of FIG. 3A) and with the M probe. Between
the two hybridizations the filter was dehybridized by washing with boiling
0.5% SDS solution for 2 minutes.
In Drosophila, insertions of elements like Minos can occur at many
different chromosomal sites, and are characterized by precise integration
extending through the terminal inverted repeats of the element without
transposition of any flanking plasmid DNA. The results of M-hybridized
SalI digests document that the events in the Medfly are of the same
nature. The transposon has inserted variable host DNA sites, and no
significant (>0.2 kb) flanking plasmid DNA to the right of the transposon
can be present, because this would have been signaled by the presence of a
2.9 kb band. The results also confirm that two independent events have
occurred in cage 3, one represented by lines 3.1 and 3.3 and the other by
lines 3.2 and 3.6 (cf. Table 3). These conclusions were also confirmed
with HincII digests. Similarly, blots of HincII digests hybridized with
the W probe showed the two endogenous w gene bands, plus a third novel
band that is characteristic of the insertion event (3.1/3.3 or 3.2/3.6).
The shortest band is longer than the 1.9 kb band that would have been
expected if the HincII site, 0.2 kb to the right of the Minos end (see
FIG. 3A) had been present. The same HincII blot hybridized with the M
probe showed that the shortest band is longer than the 1.1 kb band that
would have been expected if plasmid sequences to the left of the
transposon were present. These results were confirmed with W-hybridized
SalI digests.
To assess the integrity of the internal part of the transposon, restriction
analysis using EcoRI was performed in three lines derived from cage 25.
DNA from strains 25.7, 25.8 and 25.9 was cut with EcoRI and hybridized
with the W and M probe sequentially. In addition to the transformants
showing non-white eye phenotypes white-eyed siblings (25.9-w, 25.8-w,
25.7-w) were included in this analysis. The results of the hybridization
with the W probe indicate that the entire 3.7 kb fragment containing the
Hsp70/w marker fusion is present in the w.sup.+ transformants.
Hybridization of the same filter with the M probe, which detects
"chimeric" end fragments, showed that lines 25.8 and 25.9 contain the
same, single insertion of the transposon. The pattern in 25.7 is
consistent with the presence of two insertions, neither identical to the
25.8/25.9 event. One of these insertions, defined by the .sup..about. 3 kb
and .sup..about. 5.5 kb bands, is also present in the white-eyed siblings
of the 25.7 flies. This, presumably, represents a "silent" insertion that
does not express the phenotype either due to an undetected lesion in the
transposon, or because the transposon has integrated into a silent
(perhaps heterochromatic) genomic region.
Restriction analysis of the transformants revealed that, as predicted by
the phenotypes (Table 3), two independent transformants were represented
among the G1 progeny of cage 3, two in cage 25, and one in cage 33 (Data
for transformants from cage 33 are not shown. The restriction patterns of
three G1's from cage 1 were identical to these of the 3.2/3.6 event.
Evidently, a G0 male present in cage 3 had mated with a G0 female of cage
1, before the G0 flies were sorted into cages.) Only one of these 5
transformants (25.7) had a second (phenotypically silent) event in the
same germ line. The different transformants from the same cages are
derived either from single or multiple G0 parents. The overall frequency
of phenotypically detectable transformation events (5/390 G0 adults) is
sufficient for producing several transformants from a single experiment
since thousands of embryos can be injected and hundreds of G0 adults can
be obtained within a week using a relatively simple experimental setup.
To confirm the presence of a single Minos insertion in transformant 3.1,
third instar larva salivary gland polytene chromosomes were prepared and
in situ hybridization were performed essentially as described previously
(Zacharopoulou, A., et al., Chromosoma, 101:448 (1992)). The 3.7 kb NotI
fragment containing the Hsp70/w minigene fusion was used as probe.
Hybridization to polytene chromosomes of salivary glands from transformed
third instar larvae confirmed the presence of single Minos insertions,
allowing their cytological localization.
Those skilled in the art will recognize or be able to ascertain, using no
more than routine experimentation, many equivalents to the specific
embodiments of the invention described herein. Such equivalents are
intended to be encompassed by the following claims.
__________________________________________________________________________
# SEQUENCE LISTING
- (1) GENERAL INFORMATION:
- (iii) NUMBER OF SEQUENCES: 12
- (2) INFORMATION FOR SEQ ID NO:1:
- (i) SEQUENCE CHARACTERISTICS:
#pairs (A) LENGTH: 1775 base
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA (genomic)
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
- ACGAGCCCCA ACCACTATTA ATTCGAACAG CATGTTTTTT TTGCAGTGCG CA - #ATGTTTAA
60
- CACACTATAT TATCAATACT ACTAAAGATA ACACATACCA ATGCATTTCG TC - #TCAAAGAG
120
- AATTTTATTC TCTTCACGAC GAAAAAAAAA GTTTTGCTCT ATTTCCAACA AC - #AACAAAAA
180
- TATGAGTAAT TTATTCAAAC GGTTTGCTTA AGAGATAAGA AAAAAGTGAC CA - #CTATTAAT
240
- TCGAACGCGG CGTAAGCTTA CCTTAATCTC AAGAAGAGCA AAACAAAAGC AA - #CTAATGTA
300
- ACGGAATCAT TATCTAGTTA TGATCTGCAA ATAATGTCAC AATACAGCAT GC - #AAAAAAAT
360
- TTTAGATTGC TGCAGATCAG TAGAAGTTTA GCAACGATGG TTCGTGGTAA AC - #CTATTTCT
420
- AAAGAAATCA GAGTATTGAT TAGGGATTAT TTTAAATCTG GAAAGACACT TA - #CGGAGATA
480
- AGCAAGCAAT TAAATTTGCC TAAGTCGTCT GTGCATGGGG TGATACAAAT TT - #TCAAAAAA
540
- AATGGGAATA TTGAAAATAA CATTGCGAAT AGAGGCCGAA CATCAGCAAT AA - #CACCCCGC
600
- GACAAAAGAC AACTGGCCAA AATTGTTAAG GCTGATCGTC GCCAATCTTT GA - #GAAATTTG
660
- GCTTCTAAGT GGTCGCAGCA ATTGGCAAAA CTGTCAAGCG AGAGTGGACG CG - #ACAAATTA
720
- AAAAGTATTG GATATGGTTT TTATAAAGTA TGTTTTGTTA TTACCTGTGC AT - #CGTACCCA
780
- ATAACTTACT CGTAATCTTA CTCGTAGGCC AAGGAAAAAC CCTTGCTTAC GC - #TTCGTCAA
840
- AAAAAGAAGC GTTTGCAATG GGCTCGGGAA AGGATGTCTT GGACTCAAAG GC - #AATAGGAT
900
- ACCATCATAT TCAGCGATGA AGCTAAATTT GATGTTAGTG TCGGCGATAC GA - #GAAAACGC
960
- GTCATCCGTA AGAGGTCAGA AACATACCAT AAAGACTGCC TTAAAAGAAC AA - #CAAAGTTT
1020
- CCTGCGAGCA CTATGGTATG GGGATGTATG TCTGCCAAAG GATTAGGAAA AC - #TTCATTTC
1080
- ATTGAAGGGA CAGTTAATGC TGAAAAATAT ATTAATATTT TACAAGATAG TT - #TGTTGCCA
1140
- TCAATACCAA AACTATCAGA TTGCGGTGAA TTCACTTTTC AGCAGGACGG AG - #CATCATCG
1200
- CACACAGCCA AGCGAACCAA AAATTGGCTG CAATATAATC AAATGGAGGT TT - #TAGATTGG
1260
- CCATCAAATA GTCCAGATCT AAGCCCAATT GAAAATATTT GGTGGCTAAT GA - #AAAACCAG
1320
- CTTCGAAATG AGCCACAAAG GAATATTTCT GACTTGAAAA TCAAGTTGCA AG - #AGATGTGG
1380
- GACTCAATTT CTCAAGAGCA TTGCAAAAAT TTGTTAAGCT CAATGCCAAA AC - #GAGTTAAA
1440
- TGCGTAATGC AGGCCAAGGG CGACGTTACA CAATTCTAAT ATTAATTAAA TT - #ATTGTTTT
1500
- AAGTATGATA GTAAATCACA TTACGCCGCG TTCGAATTAA TAGTGGTCAC TT - #TTTTCTTA
1560
- TCTCTTAAGC AAACCGTTTG AATAAATTAC TCATATTTTT GTTGTTGTTG GA - #AATAGAGC
1620
- AAAACTTTTT TTTTCGTCGT GAAGAGAATA AAATTCTCTT TGAGACGAAA TG - #CATTGGTA
1680
- TGTGTTATCT TTAGTAGTAT TGATAATATA GTGTGTTAAA CATTGCGCAC TG - #CAAAAAAA
1740
# 1775 AATA GTGGTTGGGG CTCGT
- (2) INFORMATION FOR SEQ ID NO:2:
- (i) SEQUENCE CHARACTERISTICS:
#pairs (A) LENGTH: 1775 base
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA (genomic)
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
- ACGAGCCCCA ACCACTATTA ATTCGAACAG CATGTTTTTT TTGCAGTGCG CA - #ATGTTTAA
60
- CACACTATAT TATCAATACT ACTAAAGATA ACACATACCA ATGCATTTCG TC - #TCAAAGAG
120
- AATTTTATTC TCTTCACGAC GAAAAAAAAA GTTTTGCTCT ATTTCCAACA AC - #AACAAAAA
180
- TATGAGTAAT TTATTCAAAC GGTTTGCTTA AGAGATAAGA AAAAAGTGAC CA - #CTATTAAT
240
- TCGAACGCGG CGTAAGCTTA CCTTAATCTC AAGAAGAGCA AAACAAAAGC AA - #CTAATGTA
300
- ACGGAATCAT TATCTAGTTA TGATCTGCAA ATAATGTCAC AATACAGCAT GC - #AAAAAAAT
360
- TTTAGATTGC TGCAGATCAG TAGAAGTTTA GCAACGATGG TTCGTGGTAA AC - #CTATTTCT
420
- AAAGAAATCA GAGTATTGAT TAGGGATTAT TTTAAATCTG GAAAGACACT TA - #CGGAGATA
480
- AGCAAGCAAT TAAATTTGCC TAAGTCGTCT GTGCATGGGG TGATACAAAT TT - #TCAAAAAA
540
- AATGGGAATA TTGAAAATAA CATTGCGAAT AGAGGCCGAA CATCAGCAAT AA - #CACCCCGC
600
- GACAAAAGAC AACTGGCCAA AATTGTTAAG GCTGATCGTC GCCAATCTTT GA - #GAAATTTG
660
- GCTTCTAAGT GGTCGCAGCA ATTGGCAAAA CTGTCAAGCG AGAGTGGACG CG - #ACAAATTA
720
- AAAAGTATTG GATATGGTTT TTATAAAGTA TGTTTTGTTA TTACCTGTGC AT - #CGTACCCA
780
- ATAACTTACT CGTAATCTTA CTCGTAGGCC AAGGAAAAAC CCTTGCTTAC GC - #TTCGTCAA
840
- AAAAAGAAGC GTTTGCAATG GGCTCGGGAA AGGATGTCTT GGACTCAAAG GC - #AATGGGAT
900
- ACCATCATAT TCAGCGATGA AGCTAAATTT GATGTTAGTG TCGGCGATAC GA - #GAAAACGC
960
- GTCATCCGTA AGAGGTCAGA AACATACCAT AAAGACTGCC TTAAAAGAAC AA - #CAAAGTTT
1020
- CCTGCGAGCA CTATGGTATG GGGATGTATG TCTGCCAAAG GATTAGGAAA AC - #TTCATTTC
1080
- ATTGAAGGGA CAGTTAATGC TGAAAAATAT ATTAATATTT TACAAGATAG TT - #TGTTGCCA
1140
- TCAATACCAA AACTATTAGA TTGCGGTGAA TTCACTTTTC AGCAGGACGG AG - #CATCATCG
1200
- CACACAGCCA AGCGAACCAA AAATTGGCTG CAATATAATC AAATGGAGGT TT - #TAGATTGG
1260
- CCATCAAATA GTCCAGATCT AAGCCCAATT GAAAATATTT GGTGGCTAAT GA - #AAAACCAG
1320
- CTTCGAAATG AGCCACAAAG GAATATTTCT GACTTGAAAA TCAAGTTGCA AG - #AGATGTGG
1380
- GACTCAATTT CTCAAGAGCA TTGCAAAAAT TTGTTAAGCT CAATGCCAAA AC - #GAGTTAAA
1440
- TGCGTAATGC AGGCCAAGGG CGACGTTACA CAATTCTAAT ATTAATTAAA TT - #ATTGTTTT
1500
- AAGTATGATA GTAAATCACA TTACGCCGCG TTCGAATTAA TAGTGGTCAC TT - #TTTTCTTA
1560
- TCTCTTAAGC AAACCGTTTG AATAAATTAC TCATATTTTT GTTGTTGTTG GA - #AATAGAGC
1620
- AAAACTTTTT TTTTCGTCGT GAAGAGAATA AAATTCTCTT TGAGACGAAA TG - #CATTGGTA
1680
- TGTGTTATCT TTAGTAGTAT TGATAATATA GTGTGTTAAA CATTGCGCAC TG - #CAAAAAAA
1740
# 1775 AATA GTGGTTGGGG CTCGT
- (2) INFORMATION FOR SEQ ID NO:3:
- (i) SEQUENCE CHARACTERISTICS:
#pairs (A) LENGTH: 1775 base
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA (genomic)
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
- ACGAGCCCCA ACCACTATTA ATTCGAACAG CATGTTTTTT TTGCAGTGCG CA - #ATGTTTAA
60
- CACACTATAT TATCAATACT ACTAAAGATA ACACATACCA ATGCATTTCG TC - #TCAAAGAG
120
- AATTTTATTC TCTTCACGAC GAAAAAAAAA GTTTTGCTCT ATTTCCAACA AC - #AACAAAAA
180
- TATGAGTAAT TTATTCAAAC GGTTTGCTTA AGAGATAAGA AAAAAGTGAC CA - #CTATTAAT
240
- TCGAACGCGG CGTAAGCTTA CCTTAATCTC AAGAAGAGCA AAACAAAAGC AA - #CTAATGTA
300
- ACGGAATCAT TATCTAGTTA TGATCTGCAA ATAATGTCAC AATACAGCAT GC - #AAAAAAAT
360
- TTTAGATTGC TGCAGATCAG TAGAAGTTTA GCAACGATGG TTCGTGGTAA AC - #CTATTTCT
420
- AAAGAAATCA GAGTATTGAT TAGGGATTAT TTTAAATCTG GAAAGACACT TA - #CGGAGATA
480
- AGCAAGCAAT TAAATTTGCC TAAGTCGTCT GTGCATGGGG TGATACAAAT TT - #TCAAAAAA
540
- AATGGGAATA TTGAAAATAA CATTGCGAAT AGAGGCCGAA CATCAGCAAT AA - #CACCCCGC
600
- GACAAAAGAC AACTGGCCAA AATTGTTAAG GCTGATCGTC GCCAATCTTT GA - #GAAATTTG
660
- GCTTCTAAGT GGTCGCAGCA ATTGGCAAAA CTGTCAAGCG AGAGTGGACG CG - #ACAAATTA
720
- AAAAGTATTG GATATGGTTT TTATAAAGTA TGTTTTGTTA TTACCTGTGC AT - #CGTACCCA
780
- ATAACTTACT CGTAATCTTA CTCGTAGGCC AAGGAAAAAC CCTTGCTTAC GC - #TTCGTCAA
840
- AAAAAGAAGC GTTTGCAATG GGCTCGGGAA AGGATGTCTT GGACTCAAAG GC - #AATGGGAT
900
- ACCATCATAT TCAGCGATGA AGCTAAATTT GATGTTAGTG TCGGCGATAC GA - #GAAAACGC
960
- GTCATCCGTA AGAGGTCAGA AACATACCAT AAAGACTGCC TTAAAAGAAC AA - #CAAAGTTT
1020
- CCTGCGAGCA CTATGGTATG GGGATGTATG TCTGCCAAAG GATTAGGAAA AC - #TTCATTTC
1080
- ATTGAAGGGA CAGTTAATGC TGAAAAATAT ATTAATATTT TACAAGATAG TT - #TGTTGCCA
1140
- TCAATACCAA AACTATCAGA TTGCGGTGAA TTCACTTTTC AGCAGGACGG AG - #CATCATCG
1200
- CACACAGCCA AGCGAACCAA AAATTGGCTG CAATATAATC AAATGGAGGT TT - #TAGATTGG
1260
- CCATCAAATA GTCCAGATCT AAGCCCAATT GAAAATATTT GGTGGCTAAT GA - #AAAACCAG
1320
- CTTCGAAATG AGCCACAAAG GAATATTTCT GACTTGAAAA TCAAGTTGCA AG - #AGATGTGG
1380
- GACTCAATTT CTCAAGAGCA TTGCAAAAAT TTGTTAAGCT CAATGCCAAA AC - #GAGTTAAA
1440
- TGCGTAATGC AGGCCAAGGG CGACGTTACA CAATTCTAAT ATTAATTAAA TT - #ATTGTTTT
1500
- AAGTATGATA GTAAATCACA TTACGCCGCG TTCGAATTAA TAGTGGTCAC TT - #TTTTCTTA
1560
- TCTCTTAAGC AAACCGTTTG AATAAATTAC TCATATTTTT GTTGTTGTTG GA - #AATAGAGC
1620
- AAAACTTTTT TTTTCGTCGT GAAGAGAATA AAATTCTCTT TGAGACGAAA TG - #CATTGGTA
1680
- TGTGTTATCT TTAGTAGTAT TGATAATATA GTGTGTTAAA CATTGCGCAC TG - #CAAAAAAA
1740
# 1775 AATA GTGGTTGGGG CTCGT
- (2) INFORMATION FOR SEQ ID NO:4:
- (i) SEQUENCE CHARACTERISTICS:
#pairs (A) LENGTH: 1779 base
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA (genomic)
- (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: join(398..75 - #1, 812..898)
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
- ACGAGCCCCA ACCACTATTA ATTCGAACAG CATGTTTTTT TTGCAGTGCG CA - #ATGTTTAA
60
- CACACTATAT TATCAATACT ACTAAAGATA ACACATACCA ATGCATTTCG TC - #TCAAAGAG
120
- AATTTTATTC TCTTCACGAC GAAAAAAAAA GTTTTGCTCT ATTTCCAACA AC - #AACAAAAA
180
- TATGAGTAAT TTATTCAAAC GGTTTGCTTA AGAGATAAGA AAAAAGTGAC CA - #CTATTAAT
240
- TCGAACGCGG CGTAAGCTTA CCTTAATCTC AAGAAGAGCA AAACAAAAGC AA - #CTAATGTA
300
- ACGGAATCAT TATCTAGTTA TGATCTGCAA ATAATGTCAC AATACAGCAT GC - #AAAAAAAT
360
#GGT AAA CCT 415TCA GTAGAAGTTT AGCAACG ATG GTT CGT
# Met Val Arg Gly Lys Pro
# 5 1
- ATT TCT AAA GAA ATC AGA GTA TTG ATT AGG GA - #T TAT TTT AAA TCT GGA
463
Ile Ser Lys Glu Ile Arg Val Leu Ile Arg As - #p Tyr Phe Lys Ser Gly
# 20
- AAG ACA CTT ACG GAG ATA AGC AAG CAA TTA AA - #T TTG CCT AAG TCG TCT
511
Lys Thr Leu Thr Glu Ile Ser Lys Gln Leu As - #n Leu Pro Lys Ser Ser
# 35
- GTG CAT GGG GTG ATA CAA ATT TTC AAA AAA AA - #T GGG AAT ATT GAA AAT
559
Val His Gly Val Ile Gln Ile Phe Lys Lys As - #n Gly Asn Ile Glu Asn
# 50
- AAC ATT GCG AAT AGA GGC CGA ACA TCA GCA AT - #A ACA CCC CGC GAC AAA
607
Asn Ile Ala Asn Arg Gly Arg Thr Ser Ala Il - #e Thr Pro Arg Asp Lys
# 70
- AGA CAA CTG GCC AAA ATT GTT AAG GCT GAT CG - #T CGC CAA TCT TTG AGA
655
Arg Gln Leu Ala Lys Ile Val Lys Ala Asp Ar - #g Arg Gln Ser Leu Arg
# 85
- AAT TTG GCT TCT AAG TGG TCG CAG ACA ATT GG - #C AAA ACT GTC AAG CGA
703
Asn Leu Ala Ser Lys Trp Ser Gln Thr Ile Gl - #y Lys Thr Val Lys Arg
# 100
- GAG TGG ACG CGA CAG CAA TTA AAA AGT ATT GG - #A TAT GGT TTT TAT AAA
751
Glu Trp Thr Arg Gln Gln Leu Lys Ser Ile Gl - #y Tyr Gly Phe Tyr Lys
# 115
- GTATGTTTTG TTATTACCTG TGCATCGTAC CCAATAACTT ACTCGTAATC TT - #ACTCGTAG
811
- GCC AAG GAA AAA CCC TTG CTT ACG CTT CGT CA - #A AAA AAG AAG CGT TTG
859
Ala Lys Glu Lys Pro Leu Leu Thr Leu Arg Gl - #n Lys Lys Lys Arg Leu
# 130
- CAA TGG GCT CGG GAA AGG ATG TCT TGG ACT CA - #A AGG CAA TAGGATACCA
908
Gln Trp Ala Arg Glu Arg Met Ser Trp Thr Gl - #n Arg Gln
135 1 - #40 1 - #45
- TCATATTCAG CGATGAAGCT AAATTTGATG TTAGTGTCGG CGATACGAGA AA - #ACGCGTCA
968
- TCCGTAAGAG GTCAGAAACA TACCATAAAG ACTGCCTTAA AAGAACAACA AA - #GTTTCCTG
1028
- CGAGCACTAT GGTATGGGGA TGTATGTCTG CCAAAGGATT AGGAAAACTT CA - #TTTCATTG
1088
- AAGGGACAGT TAATGCTGAA AAATATATTA ATATTTTACA AGATAGTTTG TT - #GCCATCAA
1148
- TACCAAAACT ATCAGATTGC GGTGAATTCA CTTTTCAGCA GGACGGAGCA TC - #ATCGCACA
1208
- CAGCCAAGCG AACCAAAAAT TGGCTGCAAT ATAATCAAAT GGAGGTTTTA GA - #TTGGCCAT
1268
- CAAATAGTCC AGATCTAAGC CCAATTGAAA ATATTTGGTG GCTAATGAAA AA - #CCAGCTTC
1328
- GAAATGAGCC ACAAAGGAAT ATTTCTGACT TGAAAATCAA GTTGCAAGAG AT - #GTGGGACT
1388
- CAATTTCTCA AGAGCATTGC AAAAATTTGT TAAGCTCAAT GCCAAAACGA GT - #TAAATGCG
1448
- TAATGCAGGC CAAGGGCGAC GTTACACAAT TCTAATATTA ATTAAATTAT TG - #TTTTAAGT
1508
- ATGATAGTAA ATCACATTAC GCCGCGTTCG AATTAATAGT GGTCACTTTT TT - #CTTATCTC
1568
- TTAAGCAAAC CGTTTGAATA AATTACTCAT ATTTTTGTTG TTGTTGGAAA TA - #GAGCAAAA
1628
- CTTTTTTTTT CGTCGTGAAG AGAATAAAAT TCTCTTTGAG ACGAAATGCA TT - #GGTATGTG
1688
- TTATCTTTAG TAGTATTGAT AATATAGTGT GTTAAACATT GCGCACTGCA AA - #AAAAACAT
1748
# 1779 GTGG TTGGGGCTCG T
- (2) INFORMATION FOR SEQ ID NO:5:
- (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 147 amino
(B) TYPE: amino acid
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: protein
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
- Met Val Arg Gly Lys Pro Ile Ser Lys Glu Il - #e Arg Val Leu Ile Arg
# 15
- Asp Tyr Phe Lys Ser Gly Lys Thr Leu Thr Gl - #u Ile Ser Lys Gln Leu
# 30
- Asn Leu Pro Lys Ser Ser Val His Gly Val Il - #e Gln Ile Phe Lys Lys
# 45
- Asn Gly Asn Ile Glu Asn Asn Ile Ala Asn Ar - #g Gly Arg Thr Ser Ala
# 60
- Ile Thr Pro Arg Asp Lys Arg Gln Leu Ala Ly - #s Ile Val Lys Ala Asp
# 80
- Arg Arg Gln Ser Leu Arg Asn Leu Ala Ser Ly - #s Trp Ser Gln Thr Ile
# 95
- Gly Lys Thr Val Lys Arg Glu Trp Thr Arg Gl - #n Gln Leu Lys Ser Ile
# 110
- Gly Tyr Gly Phe Tyr Lys Ala Lys Glu Lys Pr - #o Leu Leu Thr Leu Arg
# 125
- Gln Lys Lys Lys Arg Leu Gln Trp Ala Arg Gl - #u Arg Met Ser Trp Thr
# 140
- Gln Arg Gln
145
- (2) INFORMATION FOR SEQ ID NO:6:
- (i) SEQUENCE CHARACTERISTICS:
#pairs (A) LENGTH: 1779 base
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA (genomic)
- (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: join(398..75 - #1, 812..1480)
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
- ACGAGCCCCA ACCACTATTA ATTCGAACAG CATGTTTTTT TTGCAGTGCG CA - #ATGTTTAA
60
- CACACTATAT TATCAATACT ACTAAAGATA ACACATACCA ATGCATTTCG TC - #TCAAAGAG
120
- AATTTTATTC TCTTCACGAC GAAAAAAAAA GTTTTGCTCT ATTTCCAACA AC - #AACAAAAA
180
- TATGAGTAAT TTATTCAAAC GGTTTGCTTA AGAGATAAGA AAAAAGTGAC CA - #CTATTAAT
240
- TCGAACGCGG CGTAAGCTTA CCTTAATCTC AAGAAGAGCA AAACAAAAGC AA - #CTAATGTA
300
- ACGGAATCAT TATCTAGTTA TGATCTGCAA ATAATGTCAC AATACAGCAT GC - #AAAAAAAT
360
#GGT AAA CCT 415TCA GTAGAAGTTT AGCAACG ATG GTT CGT
# Met Val Arg Gly Lys Pro
# 5 1
- ATT TCT AAA GAA ATC AGA GTA TTG ATT AGG GA - #T TAT TTT AAA TCT GGA
463
Ile Ser Lys Glu Ile Arg Val Leu Ile Arg As - #p Tyr Phe Lys Ser Gly
# 20
- AAG ACA CTT ACG GAG ATA AGC AAG CAA TTA AA - #T TTG CCT AAG TCG TCT
511
Lys Thr Leu Thr Glu Ile Ser Lys Gln Leu As - #n Leu Pro Lys Ser Ser
# 35
- GTG CAT GGG GTG ATA CAA ATT TTC AAA AAA AA - #T GGG AAT ATT GAA AAT
559
Val His Gly Val Ile Gln Ile Phe Lys Lys As - #n Gly Asn Ile Glu Asn
# 50
- AAC ATT GCG AAT AGA GGC CGA ACA TCA GCA AT - #A ACA CCC CGC GAC AAA
607
Asn Ile Ala Asn Arg Gly Arg Thr Ser Ala Il - #e Thr Pro Arg Asp Lys
# 70
- AGA CAA CTG GCC AAA ATT GTT AAG GCT GAT CG - #T CGC CAA TCT TTG AGA
655
Arg Gln Leu Ala Lys Ile Val Lys Ala Asp Ar - #g Arg Gln Ser Leu Arg
# 85
- AAT TTG GCT TCT AAG TGG TCG CAG ACA ATT GG - #C AAA ACT GTC AAG CGA
703
Asn Leu Ala Ser Lys Trp Ser Gln Thr Ile Gl - #y Lys Thr Val Lys Arg
# 100
- GAG TGG ACG CGA CAG CAA TTA AAA AGT ATT GG - #A TAT GGT TTT TAT AAA
751
Glu Trp Thr Arg Gln Gln Leu Lys Ser Ile Gl - #y Tyr Gly Phe Tyr Lys
# 115
- GTATGTTTTG TTATTACCTG TGCATCGTAC CCAATAACTT ACTCGTAATC TT - #ACTCGTAG
811
- GCC AAG GAA AAA CCC TTG CTT ACG CTT CGT CA - #A AAA AAG AAG CGT TTG
859
Ala Lys Glu Lys Pro Leu Leu Thr Leu Arg Gl - #n Lys Lys Lys Arg Leu
# 130
- CAA TGG GCT CGG GAA AGG ATG TCT TGG ACT CA - #A AGG CAA TGG GAT ACC
907
Gln Trp Ala Arg Glu Arg Met Ser Trp Thr Gl - #n Arg Gln Trp Asp Thr
135 1 - #40 1 - #45 1 -
#50
- ATC ATA TTC AGC GAT GAA GCT AAA TTT GAT GT - #T AGT GTC GGC GAT ACG
955
Ile Ile Phe Ser Asp Glu Ala Lys Phe Asp Va - #l Ser Val Gly Asp Thr
# 165
- AGA AAA CGC GTC ATC CGT AAG AGG TCA GAA AC - #A TAC CAT AAA GAC TGC
1003
Arg Lys Arg Val Ile Arg Lys Arg Ser Glu Th - #r Tyr His Lys Asp Cys
# 180
- CTT AAA AGA ACA ACA AAG TTT CCT GCG AGC AC - #T ATG GTA TGG GGA TGT
1051
Leu Lys Arg Thr Thr Lys Phe Pro Ala Ser Th - #r Met Val Trp Gly Cys
# 195
- ATG TCT GCC AAA GGA TTA GGA AAA CTT CAT TT - #C ATT GAA GGG ACA GTT
1099
Met Ser Ala Lys Gly Leu Gly Lys Leu His Ph - #e Ile Glu Gly Thr Val
# 210
- AAT GCT GAA AAA TAT ATT AAT ATT TTA CAA GA - #T AGT TTG TTG CCA TCA
1147
Asn Ala Glu Lys Tyr Ile Asn Ile Leu Gln As - #p Ser Leu Leu Pro Ser
215 2 - #20 2 - #25 2 -
#30
- ATA CCA AAA CTA TTA GAT TGC GGT GAA TTC AC - #T TTT CAG CAG GAC GGA
1195
Ile Pro Lys Leu Leu Asp Cys Gly Glu Phe Th - #r Phe Gln Gln Asp Gly
# 245
- GCA TCA TCG CAC ACA GCC AAG CGA ACC AAA AA - #T TGG CTG CAA TAT AAT
1243
Ala Ser Ser His Thr Ala Lys Arg Thr Lys As - #n Trp Leu Gln Tyr Asn
# 260
- CAA ATG GAG GTT TTA GAT TGG CCA TCA AAT AG - #T CCA GAT CTA AGC CCA
1291
Gln Met Glu Val Leu Asp Trp Pro Ser Asn Se - #r Pro Asp Leu Ser Pro
# 275
- ATT GAA AAT ATT TGG TGG CTA ATG AAA AAC CA - #G CTT CGA AAT GAG CCA
1339
Ile Glu Asn Ile Trp Trp Leu Met Lys Asn Gl - #n Leu Arg Asn Glu Pro
# 290
- CAA AGG AAT ATT TCT GAC TTG AAA ATC AAG TT - #G CAA GAG ATG TGG GAC
1387
Gln Arg Asn Ile Ser Asp Leu Lys Ile Lys Le - #u Gln Glu Met Trp Asp
295 3 - #00 3 - #05 3 -
#10
- TCA ATT TCT CAA GAG CAT TGC AAA AAT TTG TT - #A AGC TCA ATG CCA AAA
1435
Ser Ile Ser Gln Glu His Cys Lys Asn Leu Le - #u Ser Ser Met Pro Lys
# 325
- CGA GTT AAA TGC GTA ATG CAG GCC AAG GGC GA - #C GTT ACA CAA TTC
1480
Arg Val Lys Cys Val Met Gln Ala Lys Gly As - #p Val Thr Gln Phe
# 340
- TAATATTAAT TAAATTATTG TTTTAAGTAT GATAGTAAAT CACATTACGC CG - #CGTTCGAA
1540
- TTAATAGTGG TCACTTTTTT CTTATCTCTT AAGCAAACCG TTTGAATAAA TT - #ACTCATAT
1600
- TTTTGTTGTT GTTGGAAATA GAGCAAAACT TTTTTTTTCG TCGTGAAGAG AA - #TAAAATTC
1660
- TCTTTGAGAC GAAATGCATT GGTATGTGTT ATCTTTAGTA GTATTGATAA TA - #TAGTGTGT
1720
- TAAACATTGC GCACTGCAAA AAAAACATGC TGTTCGAATT AATAGTGGTT GG - #GGCTCGT
1779
- (2) INFORMATION FOR SEQ ID NO:7:
- (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 341 amino
(B) TYPE: amino acid
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: protein
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
- Met Val Arg Gly Lys Pro Ile Ser Lys Glu Il - #e Arg Val Leu Ile Arg
# 15
- Asp Tyr Phe Lys Ser Gly Lys Thr Leu Thr Gl - #u Ile Ser Lys Gln Leu
# 30
- Asn Leu Pro Lys Ser Ser Val His Gly Val Il - #e Gln Ile Phe Lys Lys
# 45
- Asn Gly Asn Ile Glu Asn Asn Ile Ala Asn Ar - #g Gly Arg Thr Ser Ala
# 60
- Ile Thr Pro Arg Asp Lys Arg Gln Leu Ala Ly - #s Ile Val Lys Ala Asp
# 80
- Arg Arg Gln Ser Leu Arg Asn Leu Ala Ser Ly - #s Trp Ser Gln Thr Ile
# 95
- Gly Lys Thr Val Lys Arg Glu Trp Thr Arg Gl - #n Gln Leu Lys Ser Ile
# 110
- Gly Tyr Gly Phe Tyr Lys Ala Lys Glu Lys Pr - #o Leu Leu Thr Leu Arg
# 125
- Gln Lys Lys Lys Arg Leu Gln Trp Ala Arg Gl - #u Arg Met Ser Trp Thr
# 140
- Gln Arg Gln Trp Asp Thr Ile Ile Phe Ser As - #p Glu Ala Lys Phe Asp
145 1 - #50 1 - #55 1 -
#60
- Val Ser Val Gly Asp Thr Arg Lys Arg Val Il - #e Arg Lys Arg Ser Glu
# 175
- Thr Tyr His Lys Asp Cys Leu Lys Arg Thr Th - #r Lys Phe Pro Ala Ser
# 190
- Thr Met Val Trp Gly Cys Met Ser Ala Lys Gl - #y Leu Gly Lys Leu His
# 205
- Phe Ile Glu Gly Thr Val Asn Ala Glu Lys Ty - #r Ile Asn Ile Leu Gln
# 220
- Asp Ser Leu Leu Pro Ser Ile Pro Lys Leu Le - #u Asp Cys Gly Glu Phe
225 2 - #30 2 - #35 2 -
#40
- Thr Phe Gln Gln Asp Gly Ala Ser Ser His Th - #r Ala Lys Arg Thr Lys
# 255
- Asn Trp Leu Gln Tyr Asn Gln Met Glu Val Le - #u Asp Trp Pro Ser Asn
# 270
- Ser Pro Asp Leu Ser Pro Ile Glu Asn Ile Tr - #p Trp Leu Met Lys Asn
# 285
- Gln Leu Arg Asn Glu Pro Gln Arg Asn Ile Se - #r Asp Leu Lys Ile Lys
# 300
- Leu Gln Glu Met Trp Asp Ser Ile Ser Gln Gl - #u His Cys Lys Asn Leu
305 3 - #10 3 - #15 3 -
#20
- Leu Ser Ser Met Pro Lys Arg Val Lys Cys Va - #l Met Gln Ala Lys Gly
# 335
- Asp Val Thr Gln Phe
340
- (2) INFORMATION FOR SEQ ID NO:8:
- (i) SEQUENCE CHARACTERISTICS:
#pairs (A) LENGTH: 1779 base
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: DNA (genomic)
- (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: join(398..75 - #1, 812..1480)
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
- ACGAGCCCCA ACCACTATTA ATTCGAACAG CATGTTTTTT TTGCAGTGCG CA - #ATGTTTAA
60
- CACACTATAT TATCAATACT ACTAAAGATA ACACATACCA ATGCATTTCG TC - #TCAAAGAG
120
- AATTTTATTC TCTTCACGAC GAAAAAAAAA GTTTTGCTCT ATTTCCAACA AC - #AACAAAAA
180
- TATGAGTAAT TTATTCAAAC GGTTTGCTTA AGAGATAAGA AAAAAGTGAC CA - #CTATTAAT
240
- TCGAACGCGG CGTAAGCTTA CCTTAATCTC AAGAAGAGCA AAACAAAAGC AA - #CTAATGTA
300
- ACGGAATCAT TATCTAGTTA TGATCTGCAA ATAATGTCAC AATACAGCAT GC - #AAAAAAAT
360
#GGT AAA CCT 415TCA GTAGAAGTTT AGCAACG ATG GTT CGT
# Met Val Arg Gly Lys Pro
# 5 1
- ATT TCT AAA GAA ATC AGA GTA TTG ATT AGG GA - #T TAT TTT AAA TCT GGA
463
Ile Ser Lys Glu Ile Arg Val Leu Ile Arg As - #p Tyr Phe Lys Ser Gly
# 20
- AAG ACA CTT ACG GAG ATA AGC AAG CAA TTA AA - #T TTG CCT AAG TCG TCT
511
Lys Thr Leu Thr Glu Ile Ser Lys Gln Leu As - #n Leu Pro Lys Ser Ser
# 35
- GTG CAT GGG GTG ATA CAA ATT TTC AAA AAA AA - #T GGG AAT ATT GAA AAT
559
Val His Gly Val Ile Gln Ile Phe Lys Lys As - #n Gly Asn Ile Glu Asn
# 50
- AAC ATT GCG AAT AGA GGC CGA ACA TCA GCA AT - #A ACA CCC CGC GAC AAA
607
Asn Ile Ala Asn Arg Gly Arg Thr Ser Ala Il - #e Thr Pro Arg Asp Lys
# 70
- AGA CAA CTG GCC AAA ATT GTT AAG GCT GAT CG - #T CGC CAA TCT TTG AGA
655
Arg Gln Leu Ala Lys Ile Val Lys Ala Asp Ar - #g Arg Gln Ser Leu Arg
# 85
- AAT TTG GCT TCT AAG TGG TCG CAG ACA ATT GG - #C AAA ACT GTC AAG CGA
703
Asn Leu Ala Ser Lys Trp Ser Gln Thr Ile Gl - #y Lys Thr Val Lys Arg
# 100
- GAG TGG ACG CGA CAG CAA TTA AAA AGT ATT GG - #A TAT GGT TTT TAT AAA
751
Glu Trp Thr Arg Gln Gln Leu Lys Ser Ile Gl - #y Tyr Gly Phe Tyr Lys
# 115
- GTATGTTTTG TTATTACCTG TGCATCGTAC CCAATAACTT ACTCGTAATC TT - #ACTCGTAG
811
- GCC AAG GAA AAA CCC TTG CTT ACG CTT CGT CA - #A AAA AAG AAG CGT TTG
859
Ala Lys Glu Lys Pro Leu Leu Thr Leu Arg Gl - #n Lys Lys Lys Arg Leu
# 130
- CAA TGG GCT CGG GAA AGG ATG TCT TGG ACT CA - #A AGG CAA TGG GAT ACC
907
Gln Trp Ala Arg Glu Arg Met Ser Trp Thr Gl - #n Arg Gln Trp Asp Thr
135 1 - #40 1 - #45 1 -
#50
- ATC ATA TTC AGC GAT GAA GCT AAA TTT GAT GT - #T AGT GTC GGC GAT ACG
955
Ile Ile Phe Ser Asp Glu Ala Lys Phe Asp Va - #l Ser Val Gly Asp Thr
# 165
- AGA AAA CGC GTC ATC CGT AAG AGG TCA GAA AC - #A TAC CAT AAA GAC TGC
1003
Arg Lys Arg Val Ile Arg Lys Arg Ser Glu Th - #r Tyr His Lys Asp Cys
# 180
- CTT AAA AGA ACA ACA AAG TTT CCT GCG AGC AC - #T ATG GTA TGG GGA TGT
1051
Leu Lys Arg Thr Thr Lys Phe Pro Ala Ser Th - #r Met Val Trp Gly Cys
# 195
- ATG TCT GCC AAA GGA TTA GGA AAA CTT CAT TT - #C ATT GAA GGG ACA GTT
1099
Met Ser Ala Lys Gly Leu Gly Lys Leu His Ph - #e Ile Glu Gly Thr Val
# 210
- AAT GCT GAA AAA TAT ATT AAT ATT TTA CAA GA - #T AGT TTG TTG CCA TCA
1147
Asn Ala Glu Lys Tyr Ile Asn Ile Leu Gln As - #p Ser Leu Leu Pro Ser
215 2 - #20 2 - #25 2 -
#30
- ATA CCA AAA CTA TCA GAT TGC GGT GAA TTC AC - #T TTT CAG CAG GAC GGA
1195
Ile Pro Lys Leu Ser Asp Cys Gly Glu Phe Th - #r Phe Gln Gln Asp Gly
# 245
- GCA TCA TCG CAC ACA GCC AAG CGA ACC AAA AA - #T TGG CTG CAA TAT AAT
1243
Ala Ser Ser His Thr Ala Lys Arg Thr Lys As - #n Trp Leu Gln Tyr Asn
# 260
- CAA ATG GAG GTT TTA GAT TGG CCA TCA AAT AG - #T CCA GAT CTA AGC CCA
1291
Gln Met Glu Val Leu Asp Trp Pro Ser Asn Se - #r Pro Asp Leu Ser Pro
# 275
- ATT GAA AAT ATT TGG TGG CTA ATG AAA AAC CA - #G CTT CGA AAT GAG CCA
1339
Ile Glu Asn Ile Trp Trp Leu Met Lys Asn Gl - #n Leu Arg Asn Glu Pro
# 290
- CAA AGG AAT ATT TCT GAC TTG AAA ATC AAG TT - #G CAA GAG ATG TGG GAC
1387
Gln Arg Asn Ile Ser Asp Leu Lys Ile Lys Le - #u Gln Glu Met Trp Asp
295 3 - #00 3 - #05 3 -
#10
- TCA ATT TCT CAA GAG CAT TGC AAA AAT TTG TT - #A AGC TCA ATG CCA AAA
1435
Ser Ile Ser Gln Glu His Cys Lys Asn Leu Le - #u Ser Ser Met Pro Lys
# 325
- CGA GTT AAA TGC GTA ATG CAG GCC AAG GGC GA - #C GTT ACA CAA TTC
1480
Arg Val Lys Cys Val Met Gln Ala Lys Gly As - #p Val Thr Gln Phe
# 340
- TAATATTAAT TAAATTATTG TTTTAAGTAT GATAGTAAAT CACATTACGC CG - #CGTTCGAA
1540
- TTAATAGTGG TCACTTTTTT CTTATCTCTT AAGCAAACCG TTTGAATAAA TT - #ACTCATAT
1600
- TTTTGTTGTT GTTGGAAATA GAGCAAAACT TTTTTTTTCG TCGTGAAGAG AA - #TAAAATTC
1660
- TCTTTGAGAC GAAATGCATT GGTATGTGTT ATCTTTAGTA GTATTGATAA TA - #TAGTGTGT
1720
- TAAACATTGC GCACTGCAAA AAAAACATGC TGTTCGAATT AATAGTGGTT GG - #GGCTCGT
1779
- (2) INFORMATION FOR SEQ ID NO:9:
- (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 341 amino
(B) TYPE: amino acid
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: protein
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
- Met Val Arg Gly Lys Pro Ile Ser Lys Glu Il - #e Arg Val Leu Ile Arg
# 15
- Asp Tyr Phe Lys Ser Gly Lys Thr Leu Thr Gl - #u Ile Ser Lys Gln Leu
# 30
- Asn Leu Pro Lys Ser Ser Val His Gly Val Il - #e Gln Ile Phe Lys Lys
# 45
- Asn Gly Asn Ile Glu Asn Asn Ile Ala Asn Ar - #g Gly Arg Thr Ser Ala
# 60
- Ile Thr Pro Arg Asp Lys Arg Gln Leu Ala Ly - #s Ile Val Lys Ala Asp
# 80
- Arg Arg Gln Ser Leu Arg Asn Leu Ala Ser Ly - #s Trp Ser Gln Thr Ile
# 95
- Gly Lys Thr Val Lys Arg Glu Trp Thr Arg Gl - #n Gln Leu Lys Ser Ile
# 110
- Gly Tyr Gly Phe Tyr Lys Ala Lys Glu Lys Pr - #o Leu Leu Thr Leu Arg
# 125
- Gln Lys Lys Lys Arg Leu Gln Trp Ala Arg Gl - #u Arg Met Ser Trp Thr
# 140
- Gln Arg Gln Trp Asp Thr Ile Ile Phe Ser As - #p Glu Ala Lys Phe Asp
145 1 - #50 1 - #55 1 -
#60
- Val Ser Val Gly Asp Thr Arg Lys Arg Val Il - #e Arg Lys Arg Ser Glu
# 175
- Thr Tyr His Lys Asp Cys Leu Lys Arg Thr Th - #r Lys Phe Pro Ala Ser
# 190
- Thr Met Val Trp Gly Cys Met Ser Ala Lys Gl - #y Leu Gly Lys Leu His
# 205
- Phe Ile Glu Gly Thr Val Asn Ala Glu Lys Ty - #r Ile Asn Ile Leu Gln
# 220
- Asp Ser Leu Leu Pro Ser Ile Pro Lys Leu Se - #r Asp Cys Gly Glu Phe
225 2 - #30 2 - #35 2 -
#40
- Thr Phe Gln Gln Asp Gly Ala Ser Ser His Th - #r Ala Lys Arg Thr Lys
# 255
- Asn Trp Leu Gln Tyr Asn Gln Met Glu Val Le - #u Asp Trp Pro Ser Asn
# 270
- Ser Pro Asp Leu Ser Pro Ile Glu Asn Ile Tr - #p Trp Leu Met Lys Asn
# 285
- Gln Leu Arg Asn Glu Pro Gln Arg Asn Ile Se - #r Asp Leu Lys Ile Lys
# 300
- Leu Gln Glu Met Trp Asp Ser Ile Ser Gln Gl - #u His Cys Lys Asn Leu
305 3 - #10 3 - #15 3 -
#20
- Leu Ser Ser Met Pro Lys Arg Val Lys Cys Va - #l Met Gln Ala Lys Gly
# 335
- Asp Val Thr Gln Phe
340
- (2) INFORMATION FOR SEQ ID NO:10:
- (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 5 amino
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: peptide
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
- Met Val Trp Gly Cys
1 5
- (2) INFORMATION FOR SEQ ID NO:11:
- (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 8 amino
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: peptide
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
- Trp Pro Ser Gln Ser Pro Asp Leu
1 5
- (2) INFORMATION FOR SEQ ID NO:12:
- (i) SEQUENCE CHARACTERISTICS:
#acids (A) LENGTH: 8 amino
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: peptide
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
- Trp Pro Ser Asn Ser Pro Asp Leu
1 5
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